Low-Dose Subcutaneous or Intravenous Monoclonal Antibody to Prevent Malaria.

BACKGROUND: New approaches for the prevention and elimination of malaria, a leading cause of illness and death among infants and young children globally, are needed. METHODS: We conducted a phase 1 clinical trial to assess the safety and pharmacokinetics of L9LS, a next-generation antimalarial monoclonal antibody, and its protective efficacy against controlled human malaria infection in healthy adults who had never had malaria or received a vaccine for malaria. The participants received L9LS either intravenously or subcutaneously at a dose of 1 mg, 5 mg, or 20 mg per kilogram of body weight. Within 2 to 6 weeks after the administration of L9LS, both the participants who received L9LS and the control participants underwent controlled human malaria infection in which they were exposed to mosquitoes carrying Plasmodium falciparum (3D7 strain). RESULTS: No safety concerns were identified. L9LS had an estimated half-life of 56 days, and it had dose linearity, with the highest mean (±SD) maximum serum concentration (Cmax) of 914.2±146.5 µg per milliliter observed in participants who had received 20 mg per kilogram intravenously and the lowest mean Cmax of 41.5±4.7 µg per milliliter observed in those who had received 1 mg per kilogram intravenously; the mean Cmax was 164.8±31.1 in the participants who had received 5 mg per kilogram intravenously and 68.9±22.3 in those who had received 5 mg per kilogram subcutaneously. A total of 17 L9LS recipients and 6 control participants underwent controlled human malaria infection. Of the 17 participants who received a single dose of L9LS, 15 (88%) were protected after controlled human malaria infection. Parasitemia did not develop in any of the participants who received 5 or 20 mg per kilogram of intravenous L9LS. Parasitemia developed in 1 of 5 participants who received 1 mg per kilogram intravenously, 1 of 5 participants who received 5 mg per kilogram subcutaneously, and all 6 control participants through 21 days after the controlled human malaria infection. Protection conferred by L9LS was seen at serum concentrations as low as 9.2 µg per milliliter. CONCLUSIONS: In this small trial, L9LS administered intravenously or subcutaneously protected recipients against malaria after controlled infection, without evident safety concerns. (Funded by the National Institute of Allergy and Infectious Diseases; VRC 614 ClinicalTrials.gov number, NCT05019729.).

Using infective mosquitoes to challenge monkeys with Plasmodium knowlesi in malaria vaccine studies.

BACKGROUND: When rhesus monkeys (Macaca mulatta) are used to test malaria vaccines, animals are often challenged by the intravenous injection of sporozoites. However, natural exposure to malaria comes via mosquito bite, and antibodies can neutralize sporozoites as they traverse the skin. Thus, intravenous injection may not fairly assess humoral immunity from anti-sporozoite malaria vaccines. To better assess malaria vaccines in rhesus, a method to challenge large numbers of monkeys by mosquito bite was developed. METHODS: Several species and strains of mosquitoes were tested for their ability to produce Plasmodium knowlesi sporozoites. Donor monkey parasitaemia effects on oocyst and sporozoite numbers and mosquito mortality were documented. Methylparaben added to mosquito feed was tested to improve mosquito survival. To determine the number of bites needed to infect a monkey, animals were exposed to various numbers of P. knowlesi-infected mosquitoes. Finally, P. knowlesi-infected mosquitoes were used to challenge 17 monkeys in a malaria vaccine trial, and the effect of number of infectious bites on monkey parasitaemia was documented. RESULTS: Anopheles dirus, Anopheles crascens, and Anopheles dirus X (a cross between the two species) produced large numbers of P. knowlesi sporozoites. Mosquito survival to day 14, when sporozoites fill the salivary glands, averaged only 32% when donor monkeys had a parasitaemia above 2%. However, when donor monkey parasitaemia was below 2%, mosquitoes survived twice as well and contained ample sporozoites in their salivary glands. Adding methylparaben to sugar solutions did not improve survival of infected mosquitoes. Plasmodium knowlesi was very infectious, with all monkeys developing blood stage infections if one or more infected mosquitoes successfully fed. There was also a dose-response, with monkeys that received higher numbers of infected mosquito bites developing malaria sooner. CONCLUSIONS: Anopheles dirus, An. crascens and a cross between these two species all were excellent vectors for P. knowlesi. High donor monkey parasitaemia was associated with poor mosquito survival. A single infected mosquito bite is likely sufficient to infect a monkey with P. knowlesi. It is possible to efficiently challenge large groups of monkeys by mosquito bite, which will be useful for P. knowlesi vaccine studies.

Low-dose intravenous and subcutaneous CIS43LS monoclonal antibody for protection against malaria (VRC 612 Part C): a phase 1, adaptive trial.

BACKGROUND: Human monoclonal antibodies might offer an important new approach to reduce malaria morbidity and mortality. In the first two parts of a three-part clinical trial, the antimalarial monoclonal antibody CIS43LS conferred high protection against parasitaemia at doses of 20 mg/kg or 40 mg/kg administered intravenously followed by controlled human malaria infection. The ability of CIS43LS to confer protection at lower doses or by the subcutaneous route is unknown. We aimed to provide data on the safety and optimisation of dose and route for the human antimalaria monoclonal antibody CIS43LS. METHODS: VRC 612 Part C was the third part of a three-part, first-in-human, phase 1, adaptive trial, conducted at the University of Maryland, Baltimore Center for Vaccine Development and Global Health, Baltimore, MD, USA. We enrolled adults aged 18-50 years with no previous malaria vaccinations or infections, in a sequential, dose-escalating manner. Eligible participants received the monoclonal antibody CIS43LS in a single, open-label dose of 1 mg/kg, 5 mg/kg, or 10 mg/kg intravenously, or 5 mg/kg or 10 mg/kg subcutaneously. Participants underwent controlled human malaria infection by the bites of five mosquitoes infected with Plasmodium falciparum 3D7 strain approximately 8 weeks after their monoclonal antibody inoculation. Six additional control participants who did not receive CIS43LS underwent controlled human malaria infection simultaneously. Participants were followed-up daily on days 7-18 and day 21, with qualitative PCR used for P falciparum detection. Participants who tested positive for P falciparum were treated with atovaquone-proguanil and those who remained negative were treated at day 21. Participants were followed-up until 24 weeks after dosing. The primary outcome was safety and tolerability of CIS43LS at each dose level, assessed in the as-treated population. Secondary outcomes included protective efficacy of CIS43LS after controlled human malaria infection. This trial is now complete and is registered with ClinicalTrials.gov, NCT04206332. FINDINGS: Between Sept 1, 2021, and Oct 29, 2021, 47 people were assessed for eligibility and 31 were enrolled (one subsequently withdrew and was replaced) and assigned to receive doses of 1 mg/kg (n=7), 5 mg/kg (n=4), and 10 mg/kg (n=3) intravenously and 5 mg/kg (n=4) and 10 mg/kg (n=4) subcutaneously, or to the control group (n=8). CIS43LS administration was safe and well tolerated; no serious adverse events occurred. CIS43LS protected 18 (82%) of 22 participants who received a dose. No participants developed parasitaemia following dosing at 5 mg/kg intravenously or subcutaneously, or at 10 mg/kg intravenously or subcutaneously. All six control participants and four of seven participants dosed at 1 mg/kg intravenously developed parasitaemia after controlled human malaria infection. INTERPRETATION: CIS43LS was safe and well tolerated, and conferred protection against P falciparum at low doses and by the subcutaneous route, providing evidence that this approach might be useful to prevent malaria across several clinical use cases. FUNDING: National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Mosquito bisection as a variable in estimates of PCR-derived malaria sporozoite rates.

BACKGROUND: Highly sensitive polymerase chain reaction (PCR) methods offer an alternative to the light microscopy examination of mosquito salivary glands for the determination of malaria sporozoite rates in wild caught female Anopheles. Removal of mosquito abdomens is assumed to eliminate false positives caused by malaria oocyst DNA in the midgut. This assumption has not been tested with current gold standard PCR assays, and for the variety of conditions that specimens could encounter in the laboratory and field. METHODS: Laboratory Anopheles stephensi were used that had been infected with Plasmodium falciparum 6-7 days and 14 days post infection (p.i.), when oocysts only and oocysts + sporozoites, respectively, are developed. Mosquitoes were killed and immediately frozen, air dried before being frozen, or stored under humid conditions overnight before being frozen, to simulate a range of conditions in the field. Additionally, abdomens were removed anterior to, at, or posterior to the junction of the abdomen and thorax, and both portions were processed using a standard nested PCR of the small sub-unit nuclear ribosomal genes (ssrDNA) with products visualized on agarose gels. RESULTS: Overall, 4.1 % (4/97) of head + thorax samples that were 6-7 days p.i. gave apparent false positives for sporozoites, compared to 9.3 % (9/97) that were positive for abdomens. No positives (0/52) were obtained when similar specimens were bisected anterior to the junction of the thorax and abdomen, compared to 21.2 % (11/52) that were positive for posterior portions. Multiple bands were noted for positives from the 'Frozen' treatment and the rate of false negatives due to DNA degradation appears higher under the 'Humid' treatment. Reproducibility of results for the 'Frozen' treatment was 90 %. CONCLUSIONS: Despite the importance of specimen condition and the bisection step in determining sporozoite rates, little attention has been paid to them in the literature. Recommendations from this study are that: 1) care needs to be taken to reduce DNA degradation in the field; 2) mosquito abdomens be separated anterior to the junction of the thorax and abdomen; and 3) DNA sequencing of a subsample of positive results should be undertaken if possible.

Effect of temperature on laboratory reared Anopheles dirus Peyton and Harrison and Anopheles sawadwongporni Rattanarithikul and Green.

Investigations have shown that female mosquitoes with a larger body size (determined by wing length) exhibit higher feeding rates and greater fecundity relative to smaller mosquitoes. In this study, Anopheles dirus and An. sawadwongporni were reared in the laboratory at two different temperatures (23 degrees C and 30 degrees C). Effects of the rearing temperature on body size, fecundity, and larval development period were examined by measuring wing length, adult body weight at emergence, the number of eggs produced and the length of time from the first to the fourth instar. Rearing temperature had a direct effect on body size, fecundity and larval development period for both species. Mosquitoes of both species reared at 23 degrees C were larger in body size, experienced prolonged development and produced a larger clutch of eggs relative to mosquitoes reared at 30 degrees C. However, there was no temperature effect on egg hatching rate and sex ratio.

Molecular Detection and Identification of Rickettsia Species in Ticks (Acari: Ixodidae) Collected From Belize, Central America.

Little is known about tick-borne rickettsial pathogens in Belize, Central America. We tested ixodid ticks for the presence of Rickettsia species in three of the six northern and western Belizean districts. Ticks were collected from domestic animals and tick drags over vegetation in 23 different villages in November 2014, February 2015, and May 2015. A total of 2,506 collected ticks were identified to the following species: Dermacentor nitens Neumann (46.69%), Rhipicephalus sanguineus (Latreille) (19.55%), Rhipicephalus microplus (Canestrini) (19.47%), Amblyomma cajennense complex (9.74%), Amblyomma maculatum Koch (3.47%), Amblyomma ovale Koch (0.68%), Ixodes nr affinis (0.16%), Amblyomma nr maculatum (0.12%), and Amblyomma nr oblongoguttatum (0.12%). Ticks were pooled according to species, life stage (larva, nymph, or adult), and location (n = 509) for DNA extraction and screened for genus Rickettsia by quantitative real-time polymerase chain reaction (qPCR). All 42 positive pools were found to be positive for spotted fever group (SFG) Rickettsia in pools of A. cajennense complex (n = 33), A. maculatum (n = 4), A. nr maculatum (n = 1), A. ovale (n = 1), R. sanguineus (n = 1), and I. nr affinis (n = 2). Rickettsia amblyommatis was identified from A. cajennense complex and A. nr maculatum. Rickettsia parkeri was found in A. maculatum, and Rickettsia sp. endosymbiont was detected in I. nr affinis. The presence of infected ticks suggests a risk of tick-borne rickettsioses to humans and animals in Belize. This knowledge can contribute to an effective tick management and disease control program benefiting residents and travelers.

Effects of Preexposure to DEET on the Downstream Blood-Feeding Behaviors of Aedes aegypti (Diptera: Culicidae) Mosquitoes.

Mosquito behavior is heavily influenced by the chemical molecules in the environment. This knowledge can be used to modify insect behaviors; particularly to reduce vector-host contact as a powerful method for disease prevention. N,N-Diethyl-meta-toluamide (DEET) is the most widely used insect repellent in the market and an excellent example of a chemical that has been used to modify insect behavior for disease prevention. However, genetic insensitivity and habituation in Aedes aegypti (L.) mosquitoes after preexposure to DEET have been reported. In this study, we investigated the effect of preexposure to DEET on the downstream blood-feeding behavior of Ae. aegypti mosquitoes and the duration of the effect. We exposed mosquitoes to four different DEET concentrations: 0.10, 0.12, 0.14, and 0.16% for 10 min then allowed the mosquitoes to blood-feed on an artificial blood-feeding system either immediately or after being held for 1, 3, 6, or 24 h following DEET exposure. We found that preexposing Ae. aegypti mosquitoes to 0.14 or 0.16% DEET lowered their blood engorgement level, but did not alter their landing and probing behavior when compared to the control test populations. The reduction in complete blood-feeding was observed at all time periods tested, but was only statistically significant at 3 and 6 h after the preexposure process. Because reduction in blood meal has been associated with increased refeeding, future studies analyzing the effect of this behavior using arbovirus-infected mosquitoes are needed to address the concern of potentially increased vectorial capacity.

Dengue Virus-1 Infection Did Not Alter the Behavioral Response of Aedes aegypti (Diptera: Culicidae) to DEET.

No licensed vaccine or antiviral drug against dengue virus (DENV) is available; therefore, most of the effort to prevent this disease is focused on reducing vector-host interactions. One of the most widely accepted methods of blocking vector-human contact is to use insect repellents to interfere with mosquito host-seeking behavior. Some arboviruses can replicate in the nervous system of the vector, raising the concern that arboviral infection may alter the insect behavioral response toward chemical stimuli. Three different Aedes aegypti (L.) mosquito cohorts: DENV-1-injected, diluent-injected, and uninjected were subjected to behavioral tests using a high-throughput screening system with 2.5% DEET and 0.14% DEET on 1, 4, 7, 10, 14, and 17 d postinjection. All test cohorts exhibited significant contact irritant or escape responses when they were exposed to 2.5% or 0.14% DEET. However, we found no biologically relevant irritancy response change in DENV-1-infected Ae. aegypti mosquitoes when they were exposed to DEET. Further studies evaluating the effects of other arboviral infections on insect repellents activity are necessary in order to provide better recommendation on the prevention of vector-borne disease transmission.

Detection of Dengue Virus in Mosquito Extracts and Human Clinical Samples Using a Field Expedient Molecular Platform.

Dengue fever occurs in localized outbreaks and can significantly erode troop strength and mission readiness. Timely identification of dengue virus (DENV) provides for rapid and appropriate patient management decisions, such as medical evacuation and supportive therapies, as well as help to promote Force Health Protection through vector control and personal protective measures. The "Ruggedized" Advanced Pathogen Identification Device is a field-friendly PCR (Polymerase Chain Reaction) platform that can be used to facilitate early identification of DENV. We developed a dry-format PCR assay on this platform. The assay demonstrated 100% analytical specificity for detecting dengue using a cross-reactivity panel. We used a panel of 102 acute, DENV isolation positive serum samples and 25 DENV negative samples; the assay demonstrated a clinical sensitivity of 97.1% (95% C.I. 91.6-99.4%) and specificity of 96.0% (95% C.I. 79.7-99.9%) in identifying patients with dengue infection. We also used the assay to test mosquito homogenates from 28 adult female Aedes aegypti. A single DENV infected mosquito was identified using the PCR assay and confirmed using immunofluorescence as a reference method. Much of the testing was performed under austere field conditions. Together, our results demonstrate the utility of this assay for detecting DENV in vector and human samples in field environments.

Transgenic parasites stably expressing full-length Plasmodium falciparum circumsporozoite protein as a model for vaccine down-selection in mice using sterile protection as an endpoint.

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.